1066 ONCOLOGY LETTERS 19: 1066-1073, 2020
Clinical significance of chromatin remodeling factor CHD5 expression in gastric cancer
Tadayoshi Hashimoto1, Yukinori Kurokawa1, Noriko Wada1, Tsuyoshi Takahashi1, Yasuhiro Miyazaki1, Koji Tanaka1, Tomoki Makino1, Makoto Yamasaki1, Kiyokazu Nakajima1, Masaki Mori2 and Yuichiro Doki1
1Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Osaka 565‑0871; 2Department of Surgery and Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812‑8582, Japan
Received June 3, 2019; Accepted October 8, 2019
DOI: 10.3892/ol.2019.11138
Abstract. Chromodomain helicase DNA‑binding 5 (CHD5), Introduction which is a member of the CHD family, has been identified as a tumor suppressor gene in a variety of malignancies. The Gastric cancer remains one of the leading causes of aim of the current study was to clarify the clinical significance cancer‑related deaths worldwide, and the clinical prognosis of of CHD5 expression in gastric cancer. CHD5 expression advanced gastric cancer remains poor despite multiple treat- was evaluated using immunohistochemistry (IHC) in 154 ment options (1‑4). Against this background, various studies specimens resected from patients with gastric cancer from have been performed to identify useful biomarkers to predict January 2011 to December 2013, and assessed its relationships gastric cancer prognosis (5‑7). However, unlike HER2, few with clinicopathological characteristics and survival. In vitro markers are generally used in clinical practice, and further cell proliferation, invasion, and migration assays and western investigations of novel markers are warranted. blotting analysis were performed to clarify the role of CHD5 The chromodomain helicase DNA binding 5 (CHD5) gene in human gastric cancer cell lines. Of a total of 154 patients, was first identified because of its distinctive location on the 57 (37.0%) exhibited low CHD5 expression, which was short arm of chromosome 1p36 in a region of frequent deletion significantly associated with positive lymphatic invasion in neuroblastomas (8,9). CHD5 is the fifth of nine members of (P=0.032), advanced pT status (P=0.011), and advanced pStage the CHD family, which is considered to epigenetically regulate (P=0.014). Overall survival (OS) in patients with low CHD5 chromatin organization and DNA transcription, translation, expression was significantly worse compared with patients and replication (10,11). CHD5 has been recognized as a with high CHD5 expression (hazard ratio, 1.96; 95% confi- tumor suppressor gene in various malignancies. It has been dence interval, 1.09‑3.45; log‑rank P=0.023). Cox multivariate suggested that CHD5 is frequently down‑regulated in tumor analysis for OS revealed that CHD5 expression was an inde- cells through promoter hypermethylation, and its decreased pendent prognostic factor with age and pN status. In vitro, the expression is associated with unfavorable clinical features and upregulation of CHD5 in gastric cancer cells with low CHD5 poor outcomes (12‑14). expression significantly decreased cell proliferation, migra- Regarding gastric cancer, several studies investigated the tion and invasion. CHD5 was associated with the regulation methylation status of CHD5 in gastric cancer cell lines and of multiple cancer‑related targets, including p53 and enhancer tissue samples and revealed that CHD5 was down‑regulated of zeste homolog 2 (EZH2) in western blotting analysis. In by promoter hypermethylation (15). However, only a few conclusion, since CHD5 regulated multiple cancer‑related studies have investigated the clinical implications of CHD5 targets, its expression may be a useful prognostic biomarker in protein expression in gastric cancer (16), and the mechanism patients with gastric cancer. of CHD5 function in gastric cancer has not yet been clarified. In the present study, we used immunohistochemistry (IHC) to investigate the association between CHD5 expression in tissue samples and clinicopathological characteristics in patients with gastric cancer, and clarified the mechanisms underlying Correspondence to: Dr Yukinori Kurokawa, Department of the role of CHD5 in tumor progression in gastric cancer cells Gastroenterological Surgery, Osaka University Graduate School of Medicine, 2‑2‑E2 Yamadaoka, Suita, Osaka 565‑0871, Japan in vitro. E‑mail: [email protected]‑u.ac.jp Materials and methods Key words: gastric cancer, chromatin remodeling factor, chromodomain helicase DNA‑binding 5, immunohistochemistry, Patients and tissue samples. We collected the data of 154 consec- biomarker utive patients with cT2‑4 gastric cancer between January 2011 and December 2013. Patients who received neoadjuvant chemo- therapy or underwent non‑curative resection (R2) were excluded. HASHIMOTO et al: Prognostic value of CHD5 in gastric cancer 1067
All tumors were histologically diagnosed as adenocarcinoma of used to up‑regulate CHD5. CHD5 CRISPR Activation the stomach. A total of 154 gastric cancer tissue samples were Plasmid (CHD5‑plasmid) and Control CRISPR Activation analyzed after written informed consent was obtained from each Plasmid (Control‑plasmid) for negative control (Santa Cruz patient. We used the 14th edition of the Japanese classification of Biotechnology, Inc.) were used based on the manufacturer's gastric carcinoma to determine the pathological stage (17). The protocol. present study was approved by the Institutional Review Board of Osaka University Hospital (approval number: 18227). Proliferation assay. A proliferation assay was performed using AGS, MKN45, and NUGC‑3 gastric cancer cells. Cells Cell lines and cell culture conditions. We used six gastric were seeded and incubated for 24 h in 96‑well plates, and then cancer cell lines, as follows: AGS, KATO III, MKN45, transfected with CHD5‑plasmid and Control‑plasmid. After NCI‑N87, NUGC‑3, and OCUM‑1. A human cervical cancer transfection, the 3‑(4,5‑dimethylthiazol‑2yl)‑2,5‑diphenyl cell line, HeLa, was used as the positive control. AGS, MKN45, tetrazolium bromide (MTT) assay (Sigma‑Aldrich) was OCUM‑1, NCI‑N87, and NUGC‑3 were cultured in Roswell used to estimate the cell number every 24 h until cells were Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque), confluent. HeLa in Dulbecco's modified eagle's medium (DMEM) (Wako Pure Chemical Industries), and KATO III in mixed RPMI 1640 Migration assay. A wound‑healing assay was performed using and DMEM (1:1), each containing 10% fetal bovine serum (FBS; AGS, MKN45, and NUGC‑3 gastric cancer cells. Cells trans- Thermo Fisher Scientific, Inc.). To induce cell growth, all cell fected with CHD5‑plasmid or Control‑plasmid were seeded lines were incubated in a humidified atmosphere under 5% CO2 and incubated until confluent. Cell monolayers were scratched at 37˚C . and then incubated in medium with 0.5% FBS. Every 12 h after scratching, we evaluated the percent reduction of the Immunohistochemical staining of CHD5. We prepared scratched area using the scientific image‑analysis program, 3.5‑µm‑thick sections of the resected specimens from ImageJ (National Institutes of Health, Bethesda). formalin‑fixed paraffin‑embedded blocks. These were depa- raffinized with xylene and then rehydrated with multistep Invasion assay. We used a Corning BioCoat Matrigel Invasion descending concentrations of ethanol. The sections were Chamber (Discovery Labware) to evaluate the invasion abili- autoclaved in citrate buffer at 115˚C for 20 min, immersed ties of AGS and NUGC‑3 gastric cancer cells (20). Cells were in 0.3% hydrogen peroxide to block endogenous peroxidase, cultured in serum‑free medium and then transferred to wells and incubated in horse serum for 20 min to avoid nonspecific filled with medium containing 10% FBS. After incubation for staining. The slides were incubated with monoclonal anti- 24 h cells that invaded from the culture site to the opposite side body against CHD5 (sc‑271248; dilution, 1:500; Santa Cruz of the membrane were fixed and stained with Diff‑Quick, and Biotechnology, Inc.) overnight at 4˚C, with ABC peroxidase then counted with a microscope. (Vector Laboratories) for 20 min, and with diaminobenzidine tetrahydrochloride for 1.5 min to visualize the reactions with Western blotting analysis. We extracted proteins from AGS, CHD5. Normal human Purkinje cells in cerebellum tissue MKN45, and NUGC‑3 gastric cancer cells. Proteins were were used as a positive control (Fig. S1). CHD5 expression resolved with SDS‑PAGE gels (Bio‑Rad Laboratories), trans- was evaluated in terms of the percentage and intensity of ferred onto PVDF membranes (Millipore), and incubated with tumor cells that stained positively for CHD5. As reported primary antibodies at 4˚C overnight. After incubation with previously, the percentage and intensity scores were defined secondary antibodies, signals were detected with the ECL as follows: percentage; 0%, score 0; 1‑10%, score 1; 11‑50%, Prime Western Blotting Detection reagent (GE Healthcare). score 2; 51‑100%, score 3; and intensity; negative, score 0; The following antibodies (all at 1:1,000 dilution) were used in weak, score 1; moderate, score 2; strong, score 3 (18,19). The the present study: ACTB (Sigma‑Aldrich), CHD5 (Santa Cruz final score was estimated by multiplying both scores and was Biotechnology, Inc.), p53 (DAKO), Murine Double Minute 2 classified into two groups: the high CHD5 expression group (MDM2; Santa Cruz Biotechnology, Inc.), Enhancer of zeste (>3) and the low CHD5 expression group (≤3). Representative homologue 2 (EZH2; Invitrogen), and tri‑methylation at lysine IHC staining in each group can be found in Fig. 1. 27 of histone H3 (H3K27me3; Abcam).
RNA extraction and quantitative real‑time reverse transcrip‑ Statistical analysis. We compared clinicopathological factors tion PCR (RT‑PCR). Total RNA was extracted from cells using the Chi‑squared test for categorical variables and the and converted to complementary DNA (cDNA). Primers for Mann‑Whitney U test for continuous variables. Overall RT‑PCR were as follows: CHD5 forward, CCAGTGGGCACC survival (OS) was defined as the time from the date of surgery GAGGAG, and CHD5 reverse, CTTCTTCCGCTTCCCTTT to the date of death from any cause. Recurrence‑free survival AC. GAPDH was used as an internal control. RT‑PCR was (RFS) was defined as the time from the date of surgery to carried out with THUNDERBIRD SYBR qPCR Mix (Toyobo either the date of recurrence or death from any cause. OS and Life Science) and the LightCycler 2.0 Instrument (Roche Life RFS were estimated by the Kaplan‑Meier method and tested Science). with the log‑rank test. Cox proportional hazards models were used for both univariate and multivariate analyses. A value of CHD5 up‑regulation with the CRISPR/Cas9 system. The P<0.05 was considered to indicate a statistically significant clustered regularly interspaced short palindromic repeats difference. All statistical analyses were performed using the (CRISPR)/CRISPR‑associated protein (Cas9) system was SPSS statistics software program, version 22 (IBM Corp). 1068 ONCOLOGY LETTERS 19: 1066-1073, 2020
Figure 1. Representative images of CHD5 immunohistochemical staining. (A) Absence of CHD5 nuclear staining in cancer cells, intensity score 0. (B) Weak CHD5 nuclear staining in cancer cells, intensity score 1. (C) Moderate CHD5 nuclear staining in cancer cells, intensity score 2. (D) Strong CHD5 nuclear staining in cancer cells, intensity score 3. All images were obtained with an original magnification of x400. CHD5, chromodomain helicase DNA‑binding 5.
Results patients was 60.9 and 61.1 months, respectively. OS in the low CHD5 expression group was significantly shorter than that Patient clinicopathological characteristics according to CHD5 in the high CHD5 expression group (hazard ratio [HR], 1.96; expression in gastric cancer tissues. Staining with IHC demon- 95% confidence interval [CI], 1.09‑3.45; log‑rank P=0.023; strated CHD5 in cell nuclei, but also occasionally in the cytoplasm Fig. 2A). Similarly, RFS in the low CHD5 expression group or membrane. Most non‑tumor gastric epithelial cells were was significantly shorter than that in the high CHD5 expres- strongly or moderately stained with the antibody (Fig. S2). Of sion group (HR, 2.04; 95% confidence interval [CI], 1.23‑3.46; the tumor tissues, 45 (29.2%) were negatively or weakly stained, log‑rank P=0.004; Fig. 2B). whereas 109 (70.8%) were moderately or strongly stained. After A Cox multivariate analysis for OS using all considerable multiplication of the percentage scores, 97 (63.0%) and 57 confounding factors showed that CHD5 expression was an (37.0%) of 154 patients showed high and low CHD5 expression, independent prognostic factor, along with age and pathological respectively. Clinicopathological characteristics according to N status (Table II). CHD5 expression are shown in Table I. Compared to the high CHD5 expression group, a significantly higher proportion of CHD5 baseline expression and up‑regulation using the patients in the low CHD5 expression group demonstrated posi- CRISPR/Cas9 system in gastric cancer cells. The expres- tive lymphatic invasion (P=0.032) as well as more advanced pT sion of CHD5 in gastric cancer cell lines was determined by status (P=0.011) and pStage (P=0.014). There were no significant quantitative RT‑PCR. Although CHD5 was expressed in HeLa differences between the two groups in terms of other factors. cells, its expressions in six gastric cancer cell lines (AGS, KATO III, MKN45, NCI‑N87, NUGC‑3, and OCUM‑1) were Survival analysis according to CHD5 expression. The OS down‑regulated (Fig. 3A). After transfecting AGS, MKN45, and RFS at the median follow‑up durations for all censored and NUGC‑3 cells with CHD5‑plasmid, CHD5 expression HASHIMOTO et al: Prognostic value of CHD5 in gastric cancer 1069
Table I. Clinicopathologic characteristics of gastric cancer Table II. Cox multivariate analysis of overall survival. patients according to CHD5 expression. Hazard ratio CHD5 expression Characteristics (95% CI) P‑value ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Characteristics High (n=97) Low (n=57) P‑value Age (years) 0.001 <70 1 Age (years) 0.15 ≥70 2.91 (1.54‑5.50) Median (range) 71 (40‑90) 68 (30‑88) Sex 0.71 Sex 0.21 Female 1 Male 70 (72.1%) 35 (61.4%) Male 1.13 (0.59‑2.14) Female 27 (27.9%) 22 (38.6%) Histological type Histological type 0.74 Differentiated 1 0.21 Differentiated 46 (47.4%) 29 (50.9%) Undifferentiated 1.48 (0.80‑2.75) Undifferentiated 51 (52.6%) 28 (49.1%) Lymphatic invasion 0.16 Lymphatic 0.032 No 1 invasion Yes 1.77 (0.79‑3.92) yes 68 (70.1%) 49 (86.0%) Venous invasion 0.44 No 29 (29.9%) 8 (14.0%) No 1 Venous invasion 0.067 Yes 1.28 (0.69‑2.38) Yes 30 (30.9%) 26 (45.6%) Pathological T status 0.13 No 67 (69.1%) 31 (54.4%)
Figure 2. Kaplan‑Meier survival of patients with gastric cancer according to CHD5 expression. (A) Overall survival and (B) recurrence‑free survival. CHD5, chromodomain helicase DNA‑binding 5.
Figure 3. The RNA expression of CHD5 in gastric cancer cell lines was determined using reverse transcription‑quantitative PCR. (A) GAPDH was used to normalize the CHD5 expression and HeLa was used as the reference. (B) CHD5 was upregulated by the CRISPR/Cas9 system in gastric cancer cell lines AGS, NUGC‑3 and MKN45. CHD5, chromodomain helicase DNA‑binding 5. high CHD5 expression group, and CHD5 expression was an lung cancer (18,19,21,22). Meanwhile, in gastric cancer, independent prognostic factor for gastric cancer. Furthermore, few studies have investigated the protein expression of we found that in vitro up‑regulation of CHD5 with the CHD5 in tumor samples, although several studies reported CRISPR/Cas9 system suppressed the proliferation, migration, that CHD5 was down regulated through promoter hyper- and invasion abilities of gastric cancer cells. We also showed methylation and might function as a tumor suppressor in in western blotting analysis that the up‑regulation of CHD5 gastric cancer cell lines (16,23). Our results support those suppressed the transcription of multiple targets, including of previous reports and indicate that IHC of CHD5 may be MDM2, EZH2, and H3K27me3, but up‑regulated p53. Thus, clinically useful to more accurately characterize patients CHD5 is considered to be a tumor suppressor in gastric cancer with gastric cancer. and its expression in tissue samples may be a novel biomarker As for the mechanisms of CHD5 down‑regulation, many to predict prognosis in gastric cancer patients. studies revealed that CHD5 was transcriptionally silenced Previous studies reported that low CHD5 protein by DNA methylation of its promoter, silencing the remaining expression was an independent predictive marker for allele in some cancers with 1p deletion (24‑26). Indeed, it was poor prognosis in patients with a variety of malignancies reported that in various cancers, the promoter of CHD5 was , including glioma, neuroblastoma, pancreatic cancer, and more heavily methylated than those of the remaining genes in HASHIMOTO et al: Prognostic value of CHD5 in gastric cancer 1071
Figure 4. (A) Cell proliferation assay of gastric cancer cell lines AGS, NUGC‑3 and MKN45, comparing CHD5‑ and control‑transfected cells. Gastric cancer cells transfected with CHD5‑plasmid have significantly decreased proliferation at 48, 72 and 96 h compared with transfected with Control‑plasmid. A total of 8 samples were used for each experiment. (B) Scratch wound‑healing assay of gastric cancer cell lines (AGS, NUGC‑3 and MKN45), comparing CHD5‑ and control‑transfected cells. CHD5‑transfected cells have significantly less ability to heal the artificial wound than control‑transfected cells at 12, 24 and 36 h. A total of 5 samples were used for each experiment. (C) Invasion assay of gastric cancer cell lines (AGS and NUGC‑3), comparing CHD5‑ and control‑transfected cells. CHD5‑transfected cells demonstrated significantly less invasion than control‑transfected cells. Three samples were used for each experiment. *P<0.05. CHD5, chromodomain helicase DNA‑binding 5.
the CHD family (27). Also, regarding gastric cancer, CHD5 most commonly suggested to be associated with CHD5, and was reported to be down regulated through promoter hyper- EZH2, which is a primary epigenetic regulator of various tumor methylation in cell lines and tissue samples as described above. suppressor genes (30). Additionally, several studies reported In the present study, all gastric cancer cell lines, including the mutual regulation of CHD5 and EZH2 (31). We found those whose methylation status has already been examined, that CHD5 up‑regulation was associated with up‑regulation showed suppressed CHD5 expression, with promoter methyla- of p53 and down‑regulation of EZH2. Based on these results, tion speculated to be the cause. we hypothesize that down‑regulation of CHD5 might promote Additionally, the functional role of CHD5 has been gastric cancer tumorigenesis by down‑regulating p53, one of explored in several malignancies and it has been characterized the most important tumor suppressor genes, and up‑regulating as a tumor suppressor gene, although the detailed mechanism EZH2, thereby suppressing various tumor suppressor genes. remains unclear. First, Bagchi et al suggested that CHD5 The present study has several limitations. First, it was a regulated proliferation, apoptosis, and senescence through the retrospective study conducted at a single institution. However, p19(Arf)‑p53 tumor suppressor pathway in a mouse model (12). we collected the data of consecutive patients, and we therefore In pancreatic cancer, Hall et al showed that low CHD5 expres- believe that the issue of selection bias was minimized. Second, sion prolonged cancer survival by activating the DNA damage we only assessed CHD5 in terms of proliferation, inhibitory response, and was a poor prognostic factor in patients with migration, and invasion. Further research is needed to yield pancreatic cancer treated with adjuvant chemotherapy (19). more detailed findings about the roles of CHD5. Third, we Furthermore, in renal cell carcinoma, Du et al. proposed that used western blotting to examine the expression levels of only CHD5 might epigenetically down‑regulate the expression of a limited number of cancer‑related genes and tumor suppressor various cancer‑related targets, such as oncogenes, epigenetic genes. In the future, it may be necessary to investigate other genes, epithelial‑mesenchymal transition factors, and stem cell pathways and determine the precise mechanisms of CHD5 in markers, by binding to their promoter areas (28). In addition, gastric cancer. several recent studies reported that some kinds of microRNA To the best of our knowledge, this is the first study to represented a potential epigenetic mechanism to regulate CHD5 investigate the clinical implications of CHD5 expression expression (16,29). Against this background, we used western and its functions as a tumor suppressor in gastric cancer. In blotting analysis to examine the p53 pathway, which has been conclusion, our study suggested that CHD5 expression affects 1072 ONCOLOGY LETTERS 19: 1066-1073, 2020 cancer malignancy and prognosis by regulating oncogenes and 6. Bartley AN and Hamilton SR: Select biomarkers for tumors of the gastrointestinal tract: Present and future. Arch Pathol Lab epigenetic modifiers in gastric cancer. CHD5 might function Med 139: 457‑468, 2015. as a tumor suppressor, and assessing its expression using IHC 7. Hashimoto T, Kurokawa Y, Takahashi T, Miyazaki Y, Tanaka K, may be a useful prognostic biomarker in gastric cancer. Makino T, Yamasaki M, Nakajima K, Ikeda JI, Mori M and Doki Y: Predictive value of MLH1 and PD‑L1 expression for prognosis and response to preoperative chemotherapy in gastric Acknowledgements cancer. Gastric Cancer 22: 785‑792, 2019 8. Thompson PM, Gotoh T, Kok M, White PS and Brodeur GM: CHD5, a new member of the chromodomain gene family, is Not applicable. preferentially expressed in the nervous system. Oncogene 22: 1002‑1011, 2003. Funding 9. Okawa ER, Gotoh T, Manne J, Igarashi J, Fujita T, Silverman KA, Xhao H, osse YP, White PS and Brodeur GM: Expression and sequence analysis of candidates for the 1p36.31 tumor suppressor No funding was received. gene deleted in neuroblastomas. Oncogene 27: 803‑810, 2008. 10. Kolla V, Zhuang T, Higashi M, Naraparaju K and Brodeur GM: Role of CHD5 in human cancers: 10 years later. Cancer Res 74: Availability of data and materials 652‑658, 2014. 11. Oliver SS, Musselman CA, Srinivasan R, Svaren JP, Kutateladze TG The datasets used and/or analyzed during the current study are and Denu JM: Multivalent recognition of histone tails by the PHD fingers of CHD5. Biochemistry 51: 6534‑6544, 2012. available from the corresponding author on reasonable request. 12. Bagchi A, Papazoglu C, Wu Y, Capurso D, Brodt M, Francis D, Bredel M, Vogel H and Mills AA: CHD5 is a tumor suppressor at Authors' contributions human 1p36. Cell 128: 459‑475, 2007. 13. Mulero‑Navarro S and Esteller M: Chromatin remodeling factor CHD5 is silenced by promoter CpG island hypermethylation in TH, YK, NW, and YD conceived and designed the study. TH human cancer. Epigenetics 3: 210‑215 2008. and YK analyzed the data and wrote the manuscript. TT, YM, 14. Baykara O, Tansarikaya M, Bulut P, Demirkaya A and Buyru N: CHD5 is a potential tumor suppressor in non small cell lung KT, TM, MY, KN and MM interpreted the data, were involved cancer (NSCLC). Gene 618: 65‑68, 2017. in drafting the manuscript and revising it critically for impor- 15. Wang X, Lau KK, So LK and Lam YW: CHD5 is down‑regulated tant intellectual content. All authors read and approved the through promoter hypermethylation in gastric cancer. J Biomed Sci 16: 95, 2009. final manuscript and agree to be accountable for all aspects of 16. Xu G, Zhu H, Zhang M and Xu J: Histone deacetylase 3 is asso- the research in ensuring that the accuracy or integrity of any ciated with gastric cancer cell growth via the miR‑454‑mediated part of the work are appropriately investigated and resolved. targeting of CHD5. Int J Mol Med 41: 155‑163, 2018. 17. Japanese Gastric Cancer Association: Japanese classification of gastric carcinoma: 3rd English edition. Gastric Cancer 14: Ethics approval and consent to participate 101‑112, 2011. 18. Wang L, He S, Tu Y, Ji P, Zong J, Zhang J, Feng F, Zhao J, Gao G and Zhang Y: Downregulation of chromatin remodeling factor The present study was approved by the Institutional Review Board CHD5 is associated with a poor prognosis in human glioma. of Osaka University Hospital (approved no. 18227), and it conforms J Clin Neurosci 20: 958‑963, 2013. to the provisions of the Declaration of Helsinki. Informed consent 19. Hall WA, Petrova AV, Colbert LE, Hardy CW, Fisher SB, Saka B, Shelton JW, Warren MD, Pantazides BG, Gandhi K, et al: Low was obtained from all individuals in the current study. CHD5 expression activates the DNA damage response and predicts poor outcome in patients undergoing adjuvant therapy Patient consent for publication for resected pancreatic cancer. Oncogene 33: 5450‑5456, 2013. 20. Yamamoto M, Takahashi T, Serada S, Sugase T, Tanaka K, Miyazaki Y, Makino T, Kurokawa Y, Yamasaki M, Nakajima K, et al: Not applicable. Overexpression of leucine‑rich α2‑glycoprotein‑1 is a prognostic marker and enhances tumor migration in gastric cancer. Cancer Sci 108: 2052‑2060, 2017. Competing interests 21. Garcia I, Mayol G, Rodriguez E, Suñol M, Gershon TR, Ríos J, Cheung NK, Kieran MW, George RE, Perez‑Atayde AR, et al: The authors declare that they have no competing interests. Expression of the neuron‑specific protein CHD5 is an inde- pendent marker of outcome in neuroblastoma. Mol Cancer 9: 277, 2010. References 22. Zhao R, Yan Q, Lv J, Huang H, Zheng W, Zhang B and Ma W: CHD5, a tumor suppressor that is epigenetically silenced in lung 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet‑Tieulent J and Jemal A: cancer. Lung Cance 76: 324‑331, 2012. Global cancer statistics, 2012. CA Cancer J Clin 65: 87‑108, 2015. 23. Qu Y, Dang S and Hou P: Gene methylation in gastric cancer. Clin Chim Acta 424: 53‑65, 2013. 2. Kurokawa Y, Doki Y, Mizusawa J, Terashima M, Katai H, 24. Wu X, Zhu Z, Li W, Fu X, Su D, Fu L, Zhang Z, Luo A, Sun X, Yoshikawa T, Kimura Y, Takiguchi S, Nishida Y, Fukushima N, et al: Fu L and Dong JT: Chromodomain helicase DNA binding Bursectomy versus omentectomy alone for resectable gastric protein 5 plays a tumor suppressor role in human breast cancer. cancer (JCOG1001): A phase 3, open‑label, randomised controlled Breast Cancer Res 14: R73, 2012. trial. Lancet Gastroenterol Hepatol 3: 460‑468, 2018. 25. Fatemi M, Paul TA, Brodeur GM, Shokrani B, Brim H 3. Colvin H, Mizushima T, Eguchi H, Takiguchi S, Doki Y and and Ashktorab H: Epigenetic silencing of CHD5, a novel Mori M: Gastroenterological surgery in Japan: The past, the tumor‑suppressor gene, occurs in early colorectal cancer stages. present and the future. Ann Gastroenterol Surg 1: 5‑10, 2017. Cancer 120: 172‑180, 2014. 4. Hashimoto T, Kurokawa Y, Mori M, et alUpdate on the Treatment 26. Wong RR, Chan LK, Tsang TP, Lee CW, Cheung TH, Yim SF, of gastric cancer. JMA J 1: 40‑9, 2018. Siu NS, Lee SN, Yu MY, Chim SS, et al: CHD5 downregulation 5. Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, associated with poor prognosis in epithelial ovarian cancer. Sawaki A, Lordick F, Ohtsu A, Omuro Y, Satoh T, et al: Gynecol Obstet Invest 72: 203‑207, 2011. Trastuzumab in combination with chemotherapy versus chemo- 27. Fujita T, Igarashi J, Okawa ER, Gotoh T, Manne J, Kolla V, therapy alone for treatment of HER2‑positive advanced gastric or Kim J, Zhao H, Pawel BR, London WB, et al: CHD5, a tumor gastro‑oesophageal junction cancer (ToGA): A phase 3, openlabel, suppressor gene deleted from 1p36.31 in neuroblastomas. J Natl randomized controlled trial. Lancet 376: 687‑697, 2010. Cancer Inst 100: 940‑949, 2008. HASHIMOTO et al: Prognostic value of CHD5 in gastric cancer 1073
28. Du Z, Li L, Huang X, Jin J, Huang S, Zhang Q and Tao Q: The 30. Kim KH and Roberts CW: Targeting EZH2 in cancer. Nat epigenetic modifier CHD5 functions as a novel tumor suppressor Med 22: 128‑134, 2016. for renal cell carcinoma and is predominantly inactivated by 31. Xie CR, Li Z, Sun HG, Wang FQ, Sun Y, Zhao WX, Zhang S, promoter CpG methylation. Oncotarget 7: 21618‑216130, 2016. Zhao WX, Wang XM and Yin ZY: Mutual regulation between 29. Naraparaju K, Kolla V, Zhuang T, Higashi M, Iyer R, Kolla S, CHD5 and EZH2 in hepatocellular carcinoma. Oncotarget 6: Okawa ER, Blobel GA and Brodeur GM: Role of microRNAs 40940‑40952, 2015. in epigenetic silencing of the CHD5 tumor suppressor gene in neuroblastomas. Oncotarget 7: 15977‑15985, 2016.